title: Proteomic analysis of nuclei dissected from fixed rat brain tissue using expression microdissection.
abstract: Expression microdissection (xMD) is a high-throughput, operator-independent technology that enables the procurement of specific cell populations from tissue specimens. In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate (EVA) film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p < 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.
authors.affiliation: National Cancer Institute, NIH, Bethesda, Maryland 20892, United States.
authors.forename: A R
authors.initials: AR
authors.lastname: Blackler
authors.forename: N Y
authors.initials: NY
authors.lastname: Morgan
authors.forename: B
authors.initials: B
authors.lastname: Gao
authors.forename: L R
authors.initials: LR
authors.lastname: Olano
authors.forename: M D
authors.initials: MD
authors.lastname: Armani
authors.forename: E
authors.initials: E
authors.lastname: Romantseva
authors.forename: J W
authors.initials: JW
authors.lastname: Kakareka
authors.forename: R F
authors.initials: RF
authors.lastname: Bonner
authors.forename: S
authors.initials: S
authors.lastname: Mukherjee
authors.forename: B
authors.initials: B
authors.lastname: Xiao
authors.forename: K
authors.initials: K
authors.lastname: Tran
authors.forename: T J
authors.initials: TJ
authors.lastname: Pohida
authors.forename: M R
authors.initials: MR
authors.lastname: Emmert-Buck
authors.forename: M A
authors.initials: MA
authors.lastname: Tangrea
authors.forename: S P
authors.initials: SP
authors.lastname: Markey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000166:cytology*; D002467:Cell Nucleus / Q000378:metabolism*; D011232:Chemical Precipitation; D005557:Formaldehyde / Q000378:metabolism; D013058:Mass Spectrometry; D042282:Microdissection / Q000379:methods*; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D051381:Rats; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde